Karyological analysis of small-scaled Damascus barbel, Capoeta damascina (Valenciennes, 1842) from Tigris Basin

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1 Iranian Journal of Fisheries Sciences 15(1) Karyological analysis of small-scaled Damascus barbel, Capoeta damascina (Valenciennes, 1842) from Tigris Basin Dorafshan S. 1* ; Roozdar A. 1 Received: November 2014 Accepted: September Department of Natural Resources, Isfahan University of Technology, Isfahan, , Iran. * Corresponding author's sdorafshan@cc.iut.ac.ir Keywords: Damascus barbel, Capoeta damascina, Karyotype, Tigris Basin. Introduction Cyprinids are one of the major components of the freshwater fish diversity of Asia. Among different genera of this family; Capoeta shows wide distribution in the southwest area of this mainland. It contains about 20 species of which 8 occur in Iran (Keivany et al., 2015). Small-scaled Damascus barbel, Capoeta damascina (Valenciennes, 1842) is one of the most important species of Capoeta in Iran. It attains the greatest size and the highest density among all other Capoeta species in this region. Chromosome analysis is a valuable tool for systematic evaluation, biodiversity, conservation, stock assessment and aquaculture (Dorafshan and Kalbassi, 2006; Kalbassi et al., 2006; Pisano et al., 2007). Despite the importance of fish cytogenetics, when available data sets on fish karyotype are analysed, it is clear that they are still very incomplete (Gromicho and Collares-Pereira, 2007). In the Cyprininae subfamily, we can find evolutionary diploids (2n 48-50) e.g. smallmouth lotak, Cyprinion kais (Nasri et al., 2010), tetraploids (2n ) e.g. common carp, Cyprinus carpio (Al-Sabti, 1986) and Schizothorax zarudnyi (Kalbassi et al., 2008) and hexaploid (2n ) e.g. Barbus canis (Gorshkova et al., 2002). Changes in polyploidy level may be a key factor in the cause of evolutionary changes in Cyprinidae. Some reports are available on the karyology of different species and/or subspecies of Capoeta like C. trutta and C. capoeta ulma from Tigris River, Turkey (Kiliç- Demirokand Ünlü, 2001), and C. c. grasilis from the Caspian Sea Basin, Iran (Darestani et al., 2006). However

2 543 Dorafshan. and Roozdar, Karyological analysis of small-scaled Damascus barbell the only report available on the karyology of C. damascina is based on the Wadi Karak stream population from the Kingdom of Jordan (Gorshkova et al., 2002). The aim of this study was to investigate the karyotype of C. damascina for basic information for evaluation, conservation and/or aquaculture purposes. aterials and methods Fifteen specimens (11-17 g and 7-14 cm SL, 5 males and 10 females) of C. damascina were obtained on 15 June 2010 from the onj River 50 41' E and 31 35' N, a tributary of the Karoon River, Tigris Basin, located in the Charmahal-o-Bakhtiari Province, west of Iran. The fish were delivered live to the lab, in 100 L well-aerated aquaria at C following guidelines for treating experimental fish approved by the Isfahan University of Technology Committee. Chromosome preparation was made following the standard method of Thorgaard and Disney (1990) with some modification. Briefly, the fish received two identical intraperitoneal (i.p.) injections of phytohaemaglutinin, PHA (Baharafshan, Iran) with an interval of 24-h, final dose 40 µg/gbw. 12 h after the final PHA injection, the fish received i.p. injection of µg/g body weight of colchicine (Sigma, USA) as a mitogenic inhibitor. The head kidney and the gill filaments of each fish were extracted separately for each fish, 7-8 h after colchicine injection. The tissues were immersed in a cold (4 C) hypotonic solution of 0.1 KCl for 45 min. The suspension was centrifuged at 1300 rpm for 10 min, supernatant removed and the rest was fixed with cold-fresh Carnoy's solution (3:1 methanol and glacial acetic acid) as a fixative. Three changes of fixative were made at 30 min intervals, followed by smear preparation on cold lamella using splash method. The slides were stained by 10% Giemsa. A minimum of 4 metaphase spreads of the kidney and gill tissues were examined for each specimen using a Nikon microscope (Fujix HC-300zi, Japan) to account for the chromosome number. After chromosome number determination, the best spread was photographed using compact microscope (NTHCS, Swiss) at 4000 X to provide the karyogram. The morphometric measurements were done by Image tools V.6 software. Arm ratio (AR) expressed as the ratio of the long arm to the short arm length of each pair of chromosome. Relative length of chromosome (RL) was the absolute length of each chromosome pair divided by the sum of the absolute length of total chromosome expressed in percentage. The centromic index (CI) or form percentage (F%) calculated as the ratio of the length of the short arm of the chromosome to that of the total chromosome, ordinarily expressed as a percentage. While, r- value and total form percentage (TF%) were the ratio between the shortest to the longest chromosome pair and the

3 Iranian Journal of Fisheries Sciences 15(1) ratio of the length of the short arm of the total chromosome to the total length of all chromosome respectively (Levan et al.,1964; acgregor and Varley, 1993). Chromosomes were classified into metacentric (), submetacentric (S), subtelocentric () and acrocentric (A) based on the Levan et al. (1964) recommendation when the AR were in the range of 1-1.7, 1.7-3, 3-7 and >7, respectively. The karyogram and ideogram were provided using Adobe Photoshop 6.0 and icrosoft Excel 2003 respectively. Results and discussion The count of chromosomes ranged from 147 to 152 per metaphases, with a mode of 150 representing 67% of the metaphases (Table 1). The sizes of the chromosomes were in the range of µm. The largest and smallest chromosomes were a pair of S and A, respectively. The long arm and short arm ranges were and µm, respectively (Table 2). The ranges of AR, RL and CI or F were in the ranges of 1.08-, % and %, respectively (Table 2). The r-value and TF index were calculated as 0.37 and 24.36%, respectively. There were 9 pairs of, 30 pairs of S, 22 pairs of and 14 pairs of A chromosomes providing the chromosome number and formula of C. damascina as 2n=150 and 2n= 9+30S+22+14A (Table 2). The chromosome spread, karyogram and ideogram of C. damascina are presented in Figs. 1 to 3, respectively. The homologous pairs of chromosomes were arranged according to the classification. The NF was 228, which was calculated by assigning a value of two arms for /S chromosomes and one arm for the A/T chromosomes. No sex chromosomes were clearly observed. Fontana et al. (1997) stated the range between 2n=22-26 for Nototheniidae to 2n= in Acipensereidae. While, Hallerman (2003) reported the lowest chromosome number as 2n=16 in Sphaerichthys osphramenoides (Belontidea) to 2n=446 in Datchus dipogon. Nevertheless, it is well documented that most of the cyprinid fish have 2n=50, although some of them have higher chromosome number such as 2n= in common carp (Al-Sabti, 1986).

4 545 Dorafshan. and Roozdar, Karyological analysis of small-scaled Damascus barbell Table 1: Chromosome complement of small- scaled Damascus barbel, Capoeta damascina Valenciennes, 1842), based on observed frequency, 2n = 150 Number of fish Sex* Chromosome number Total Karytype** (2n=150) metaphases -S -A NF F F F F F F F F Immature Totals *- : ale; F: Female. **- -S: etacentric-submetacentric; -A: Subtelocentic-Acrocentric. NF: Number of Fundamental. The diploid chromosome number of C.damascina was determined from Tigris Basin for the first time and defined as 2n=150 including 18, 60 S, 44 and 28 A. In general, fish can survive and reproduce actively even with some chromosomal rearrangements which maybe pernicious to other vertebrates like mammals. Based on available information (Table 3), 2n = 150 might be acceptable as a diploid chromosome number and this genus of Cyprinidae could be categorised as hexaploid cyprinids. It has been reported that different fish species can undergo different levels of ploidy such as diploid, tetraploid and hexaploid levels which has been observed in some Cyprinids (Tsigenopoulos et al., 2002), Salmonids (Gromicho and Collares- Pereira, 2007) and Acipenserids (Fontana et al., 2007). Changing in ploidy levels can be categorised as an important speciation force in many groups of fish (Fontana et al., 2008).

5 Iranian Journal of Fisheries Sciences 15(1) Table 2: Numeral characteristics of the karyotype of small-scaled Damascus barbel. Classification * Cl c (%) RL b (%) AR a Total length (µm) Long arm (µm) Short arm (µm) Chromosome pair S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S

6 547 Dorafshan. and Roozdar, Karyological analysis of small-scaled Damascus barbell Continued Table 2: Chromosome pair Short arm Long arm Total length Ara RLb (%) Clc (%) Classification* (µm) (µm) (µm) A A A A A A A A A A A A A A a : Arm ratio, b : Relative length, c : Centromic index, d : The chromosomes (75 pairs) are classified as, metacentric; S, submetacentric;, subtelocentric; A, acrocentric; according to Levan et al. (1964). Refer to the material and methods for detailed information. Table 3: Some recent studies on karyotype of Capoeta spp. from different rivers/basins. Species River/Basin 2n Classification* NF References C. trutta Tigris River /S + 80/A 220 Demirok and Ünlü, 2001 C. capoeta umbla Tigris Rriver /S + 64/A 236 Demirok and Ünlü, 2001 C. damascina Wadi Karak Stream/Dead Sea /S A 76/S A 76/S A Gorshkova et al., 2002 C. capoeta gracilis Sefidrood River S T 234 Darestani et al., 006 /Caspian Sea C. capoeta gracilis adarsoo River /Caspian Sea S T 230 Darestani et al., 2006 C. damascina onj Rriver /Tigris S A 228 Present study *-, metacentric; S, submetacentric;, subtelocentric; A, acrocentric.

7 Iraniann Journal of Fisheries Sciences 15(1) Figure 1: Chromosome spread (2n=150) of head kidney tissue from small-scaled Damascus barbel, Capoeta damascina. Barr = 5 µm. Figure 2: Standard karyotype of small-scaled Damascus barbel, Capoeta damascina a (Valenciennes, 1842) (2n=150). 1-9 (metacentric); 10-39(submetacentric); (subtelocentric) and (acrocentric) according to Levan et al.. (1964).

8 549 Dorafshan. and Roozdar, Karyological analysis of small-scaled Damascus D barbell Figure 3: Standard ideogram of small-scaled Damascus barbel, Capoeta C damascina. The longest and shortest chromosomes are number 10 (S) and the last one o (no.75, A) with total length of 4.10 and 1.54 µm, respectively. r Other available reportss about diploid chromosome numberss of Capoeta species from different basins indicated 2n as 148 in C. damascina from the Dead Sea Basin, the kingdom of Jordan (Gorshkova et al., 2002), 2n=150 in Capoeta trutta and C. capoeta from Tigris River, Turkey (Kiliç-Demirok and Ünlü, 2001) and C. C. grasilis from the Caspian Sea Basin, Iran (Darestani et al., 2006). Polymorphismm in chromosome number as well as its classification is very common phenomena in fish (Gorshkova et al., 2002; Nasri et al., 2010; Tablee 3). These differences may be caused by evolutionary phenomena, exposure to contaminated water, hybridizationn and meiotic and mitotic disjunctions (Hartly, 1998). Chromosomal rearrangements such as pericentric inversion and Robertsonian fusions are other factors which can vindicate different chromosome classificationn and NF for the same species. Comparison of thesee data might relay close phyletic connections of Capoeta genus in different areas of the iddle East, but it is necessary to consider the chromosome numberr of other species of this genus. Because of the large number of small chromosomes in all studied Capoeta species including C. damascina, it wouldd be recommended to use other staining techniques such as G- or C-banding orr Ag-NOR. These data would be more helpful in cytotaxonomy and phylogenetic studies of Capoeta. It could be concluded that t C. damascinaa from the onj River, Tigris Basin has 2n=150 chromosome and could be categorized as a hexaploid species. However H more detailedd studies would be recommended to find out the ploidy origin and exact chromosome kind and NF. N References: Al-Sabti, K., Karyotypes of Cyprinus carpioo and Leuciscus

9 Iranian Journal of Fisheries Sciences 15(1) cephalus orientalis. Cytobios, 47, Darestani, S.P., Bazyar Lakeh, A. and Hasan-Zadeh Kiabi, B., A karyological study of Barbus capito, Barbus mursa and two populations of Capoeta capoeta from northern Iran. Iranian Journal of Natural Resource, 58, (In Farsi). Dorafshan, S. and Kalbassi,.R., Karyological study of Ctenopharyngodon idella Hypophtalmichthys nobilis F1 hybrids. Iranian Journal of Biology, 20, (In Farsi). Fontana, F., Rossi, R., Lanfredi,., Arlati, G. and Bronzi, P., Cytogenetic chracterictization of cell lines from three sturgeon species. Caryologia, 50, Fontana, F., Zane, L., Pepe, A. and Congiu, L., Polyploidy in Acipenseriformes: cytogenetic and molecular approaches. In: Pisano E, Ozouf-Costaz C, Foresti F, KapoorBG, eds. Fish cytogenetics. Science Publishers. Enfield, NH, USA, Fontana, F., Congiu, L., udrak,v.a., Quattro, J.., Smith, T.I.J., Ware, K. and Doroshov S.I., Evidence of hexaploid karyotype in shortnose sturgeon. Genome. 51(2), Gromicho,. and Collares-Pereira,.J., The evolutionary role of hybridization and polyploidy in an Iberian cyprinid fish - a cytogenetic review. In: Pisano E, Ozouf-Costaz C, Foresti F, KapoorBG, eds. Fish cytogenetics. Science Publishers. Enfield, NH, USA Hallerman, E., Population genetics: principles and applications for fisheries scientists. 1st ed. New York: American Fisheries Society. Hartley, R.W., Barnase and Barstar: two small proteins to fold and fit together.trends in Biotechnology, 14, Kalbassi,.R., Dorafshan, S., Tavakolian, T. Khazab,. and Abdolhay H., Karyological analysis of endangered Caspian salmon, Salmo trutta caspius (Kessler, 1877). Aquaculture Research, 37(13), Kalbassi,.R., Hosseini, S.V. and Tahergorbani, R., Karyotype analysis in Schizothorax zarudnyi from Hamoon Lake, Iran. Turkish Journal of Fisheries and Aquatic Science, 8, Keivany, Y., Nasri,., Abbasi, K. and Abdoli, A., Atlas of inland water fishes of Iran. Iran Department of Environment. Kiliç-Demirok, N. and Ünlü, E., Karyotypes of cyprinid fish Capoeta trutta and Capoeta capoeta umbla (Cyprinidae) from the Tigris River. Turkish Journal of Zoology, 25, Levan, A., Fredga, K. and Sandberg, A.A., A nomenclature for centromeric position on chromosomes. Heredity, 52,

10 551 Dorafshan. and Roozdar, Karyological analysis of small-scaled Damascus barbell acgregor, H. and Varley, J., Working with animal chromosomes. 1st ed. New York: John Wiley. Nasri,., Keivany, Y. and Dorafshan, S., First karyological analysis of smallmouth lotak, Cyprinion kais Heckel, 1843, an endemic cyprinid fish from Tigris-Euphrates Basin. Italian Journal of Zoology, 73, Pisano, E., Ozouf-Costaz, C., Foresti, F. and Kapoor, B.G., Fish cytogenetics. 1st ed. Enfield, NH: Science Publishers. Thorgaard, G.H. and Disney, J.E., Chromosome preparation and analysis. In: Schreck CB, oyle PB, eds. ethods for fish biology. Bethesda, D: American Fisheries Society, Tsigenopoulos, C.S., Ra b, P., Naran, D. and Berrebi, P., ultiple origins of polyploidy in the phylogeny of southern African barbs (Cyprinidae) as inferred from mtdna markers. Heredity, 88,